C3A receptor ligands

ABSTRACT

A compound according to Formula (I):                    
     wherein: 
     A represents C 1-4  alkylene, unsubstituted or optionally substituted by C 1-4  alkyl or aryl; or A forms a 5-8 membered fused aliphatic ring with the adjacent phenyl ring; 
     m is an integer from 1 to 3; 
     each R 1  is independently selected form the group consisting of halo, C 1-4  alkyl, methanesulfonyl, alkoxy, nitrile, dimethylamine, methylenedioxy and CF 3 ; and 
     R 2  is hydrogen or methyl is provided.

This application is a 371 of PCT/US99/182,564 filed Aug. 11, 1999, whichclaims the benefit of Provisional application No. 60/096,055 filed Aug.11, 1998.

FIELD OF THE INVENTION

The present invention relates to novel C3A receptor ligands,pharmaceutical compositions containing these compounds and methods ofusing the present compounds to treat inflammation.

BACKGROUND OF THE INVENTION

Anaphylatoxins are 74-77 amino acid bioactive fragments of C5, C3 and C4that are generated in vivo during complement activation. Binding of theanaphylatoxins to specific cell surface receptors initiates andmaintains the inflammatory process. The fragments are believed to elicitmast cell and basophil degranulation with release of histamine,cytokines and other inflammatory mediators and induce smooth muscle cellcontraction. They are potent inflammatory mediators, inducing cellulardegranulation, smooth muscle contraction, arachidonic acid metabolism,cytokine release, cellular chemotaxis. See Gerard, C., and Gerard, N. P.(1994) Annu. Rev. Immunol. 12, 775-808; Hugli, T. E. (1984) SpringerSemin. Immunopathol. 7, 193-219; Bitter-Suermann, D. (1988) in TheComplement System, Ed. by K. Rother & G. Till, Springer Verlag,Heidelberg 367-395.

The present fragments have been implicated in the pathogenesis of anumber of inflammatory diseases. See Vogt, W. (1986) Complement 3,177-188; Morgan, B. P. (1994) European J Clin Investigation 24, 219-228.Studies have demonstrated the presence of a C3A receptor (C3A-R) onguinea pig platelets, rat mast cells, human neutrophils, eosinophils andplatelets (Bitter-Suermann, D. (1988) in The Complement System, Ed. byK. Rother & G. Till, Springer Verlag, Heidelberg 367-395). A singleclass of high affinity C3A binding sites has been characterized on humanneutrophils and differentiated U937 cells (KIos, A., Bank, S., Gietz,C., Bautsch, W., Köhl, J., Burg, M., and Kretzschmar, T (1992)Biochemistry 31, 112741-1282). Competition binding and functionaldesensitization studies are consistent with the presence of a receptorfor C3A which is distinct from the C5A-R (Bitter-Suernann, D. (1988) inThe Complement System, Ed. by K. Rother & G. Till, Springer Verlag,Heidelberg 367-395; Klos, A., Bank, S., Gietz, C., Bautsch, W., Köhl,J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282).However, there is evidence that C3A and C4A may bind to the samereceptor as the two anaphylatoxins cross desensitize guinea pig ilealtissue (Hugli, T. E. (1984) Springer Semin. Immunopathol. 7, 193-219;Bitter-Suermann, D. (1988) in The Complement System, Ed. by K Rother &G. Till, Springer Verlag, Heidelberg 367-395), although otherinvestigators using guinea pig macrophages indicate that there may beseparate receptors (Murakami, Y., Yamamoto, T., Imamichi, T., Nagasawa,S. (1993) Immunol. Lett. 36, 301-304). Functional activity of the C 3A-Ris sensitive to pertussis toxin, consistent with the binding site beingcomposed of a GPCR (Klos, A., Bank, S., Gietz, C., Bautsch, W., köhl,J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282).

A complete understanding of the role of C3A in the pathogenesis of theinflammatory response has been hampered by the lack of the clonedreceptor. The present invention provides methods of using and functionalcharacterization of human C3A receptor. This same receptor was recentlyindependently cloned from an HL-60 library by low-stringency screeningwith a fMetLeuPhe receptor probe and, lacking functional data, claimedto be an orphan receptor (AZ3B,8). Mouse L cells expressing AZ3B failedto bind and respond to the agonists examined, although C3A was nottested (Roglic, A., Prossnitz, E. R., Cavanagh, S. L., Pan, Z, Zou, A. &Ye, R. D. (1996) Biochimica et Biophysica Acta 1305, 39-43). The presentinvention discloses compounds that antagonize C3A receptor function.

Clearly, there is a need for factors that mediate inflammation and theirroles in dysfunction and disease. There is a need, therefore, foridentification and characterization of compounds which antagonize C3Areceptor function, and which can play a role in preventing, amelioratingor correcting dysfunctions or diseases.

Thus, C3A ligands offer a unique approach towards the pharmacotherapy ofimmune and inflammatory diseases such as rheumatoid arthritis,Alzheimer's disease, psoriasis, gout, multiple sclerosis, systemic lupuserythematosus, glomerulonephritis and adult respiratory distresssyndrome.

SUMMARY OF THE INVENTION

The present invention involves compounds represented by Formula (I)hereinbelow and their use as C3A receptor ligands which are useful inthe treatment of a variety of diseases associated with complementactivation and or increased levels of anaphylatoxins, including but notlimited to rheumatoid arthritis, Alzheimer's disease, psoriasis, gout,multiple sclerosis, systemic lupus erythematosus, glomerulonephritis andadult respiratory distress syndrome.

The present invention further provides methods for antagonizing C3Areceptors in an animal, including humans, which comprises administeringto an animal in need of treatment an effective amount of a compound ofFormula (1), indicated hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention are selected from Formula (I)hereinbelow:

wherein:

A represents C₁₋₄ alkylene, unsubstituted or optionally substituted byC₁₋₄ alkyl or aryl; or

A forms a 5-8 membered fused aliphatic ring with the adjacent phenylring;

m is an integer from 1 to 3;

each R₁ is independently selected from the group consisting of halo,C₁₋₄ alkyl, methanesulfonyl, alkoxy, nitrile, dimethylamine,methylenedioxy and CF₃; and

R₂ is hydrogen or methyl.

Preferably, A represents phenethyl.

Preferably m is 0.

Preferably, R₁ represents hydrogen.

Preferably, R₂ represents hydrogen.

As used herein, “alkyl” refers to an optionally substituted hydrocarbongroup joined together by single carbon-carbon bonds. The alkylhydrocarbon group may be linear, branched or cyclic, saturated orunsaturated. Preferably, the group is linear. Preferably, the group isunsubstituted. Preferably, the group is saturated.

As used herein “cycloalkyl” refers to 3-7 membered carbocyclic rings.

As used herein “heterocycloalkyl” refers to 4-7 membered heterocyclicrings containing 1 to 2 heteroatoms selected from N, O and S.

As used herein, “aryl” refers to an optionally substituted aromaticgroup with at least one ring having a conjugated pi-electron system,containing up to two conjugated or fused ring systems. “Aryl” includescarbocyclic aryl, heterocyclic aryl and biaryl groups, all of which maybe optionally substituted. A preferred aryl group is phenyl.

As used herein “acyl” refers to alkylcarbonyl.

The compounds of the present invention may contain one or moreasymmetric carbon atoms and may exist in racemic and optically activeforms. All of these compounds and diastereomers are contemplated to bewithin the scope of the present invention.

Preferred compounds in the present invention include:

1-Naphthyloxyacetylarginine;

1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;

(2,2-Diphenylethoxy)acetylarginine;

(2,2-Diphenylethoxy)acetyl-Nα-methylarginine;

(3-Chlorobenzyloxy)acetylarginine;

2-Naphthyloxyacetylarginine;

(2,3-Dimethylphenoxy)acetylarginine;

8-(Quinolinyloxy)acetylarginine;

6-(Quinolinyloxy)acetylarginine;

2-(1-Bromonaphthyloxy)acetylarginine;

(4-Benzyloxyphenoxy)acetylarginine; and

2-(6-Methoxynaphthyloxy)acetylarginine.

More preferred compounds of the present invention include:

1-Naphthyloxyacetylarginine;

1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;

(2,2-Diphenylethoxy)acetylarginine; and

2-Naphthyloxyacetylarginine.

The most preferred compounds of the present invention include:

1-Naphthyloxyacetylarginine; and

(2,2-Diphenylethoxy)acetylarginine.

An especially preferred compound of the present invention is(2,2-Diphenylethoxy)acetylarginine.

The present compounds can also be formulated as pharmaceuticallyacceptable salts and complexes thereof. Pharmaceutically acceptablesalts are non-toxic salts in the amounts and concentrations at whichthey are administered.

Pharmaceutically acceptable salts include acid addition salts such asthose containing sulfate, hydrochloride, fumarate, maleate, phosphate,sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts canbe obtained from acids such as hydrochloric acid, maleic acid, sulfuricacid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lacticacid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamicacid, fumaric acid, and quinic acid.

Pharmaceutically acceptable salts also include basic addition salts suchas those containing benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium, anmmonium, alkylamine, and zinc, whenacidic functional groups, such as carboxylic acid or phenol are present.

The present invention provides compounds of Formula (I) above which canbe prepared using standard techniques. An overall strategy for preparingpreferred compounds described herein can be carried out as described inthis section. The examples which follow illustrate the synthesis ofspecific compounds. Using the protocols described herein as a model, oneof ordinary skill in the art can readily produce other compounds of thepresent invention.

All reagents and solvents were obtained from commercial vendors.Starting materials (e.g., amines and epoxides) were synthesized usingstandard techniques and procedures. The present invention providescompounds of formula (I) above which can be prepared using standardtechniques. An overall strategy for preparing preferred compoundsdescribed herein can be carried out as described in this section. Theexamples which follow illustrate the synthesis of specific compounds.Using the protocols described herein as a model, one of ordinary skillin the art can readily produce other compounds of the present invention.

All reagents and solvents were obtained from commercial vendors.Starting materials were synthesized using standard techniques andprocedures.

Aryloxyacetylarginines (eg. 5) can be prepared on solid phase. Anappropriately protected arginine derivative such as Fmocarginine(Boc)₂(1) is coupled to chlorotrityl resin with an amine base such asdiisopropylamine to give 2. Deprotection, and derivatization withbromoacetic acid yields the intermediate bromoacetamide 3. Reaction ofthis with arylalcohols under basic conditions such as potassiumcarbonate or amine bases in DMSO with heating yields the aryloxyacetylproduct 4. Deprotection with TFA in the presence of a cation scavengersuch as triisopropylsilane, dimethylsulfide, ethanedithiol, anisole,water, or some combination of these yields the cleaved product 5.

Alkyloxy and aryloxy derivatives can be prepared by coupling anappropriately protected arginine derivative such as Fmocarginine(Mtr)(6) to Wang resin. Deprotection and coupling with an alkyloxyacetic acidor aryloxyacetic acid yields the protected resin bound intermediate 7.Finally, deprotection with TFA in the presence of a cation scavengeryields the final product 8.

With appropriate manipulation and protection of any chemicalfunctionality, synthesis of the remaining compounds of Formula (I) isaccomplished by methods analogous to those above and to those describedin the Experimental section.

In order to use a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof for the treatment of humans and other mammals,it is normally formulated in accordance with standard pharmaceuticalpractice as a pharmaceutical composition.

The present ligands can be administered by different routes includingintravenous, intraperitoneal, subcutaneous, intramuscular, oral,topical, transdermal, or transmucosal administration. For systemicadministration, oral administration is preferred. For oraladministration, for example, the compounds can be formulated intoconventional oral dosage forms such as capsules, tablets and liquidpreparations such as syrups, elixirs and concentrated drops.

Alternatively, injection (parenteral administration) may be used, e.g.,intramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds of the invention are formulated in liquidsolutions, preferably, in physiologically compatible buffers orsolutions, such as saline solution, Hank's solution, or Ringer'ssolution. In addition, the compounds may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms canalso be produced.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration, for example, may be throughnasal sprays, rectal suppositories, or vaginal suppositories.

For topical administration, the compounds of the invention can beformulated into ointments, salves, gels, or creams, as is generallyknown in the art.

The amounts of various calcilytic compounds to be administered can bedetermined by standard procedures taking into account factors such asthe compound IC50, EC50, the biological half-life of the compound, theage, size and weight of the patient, and the disease or disorderassociated with the patient. The importance of these and other factorsto be considered are known to those of ordinary skill in the art.

Amounts administered also depend on the routes of administration and thedegree of oral bioavailability. For example, for compounds with low oralbioavailability, relatively higher doses will have to be administered.

Preferably the composition is in unit dosage form. For oral application,for example, a tablet, or capsule may be administered, for nasalapplication, a metered aerosol dose may be administered, for transdermalapplication, a topical formulation or patch may be administered and fortransmucosal delivery, a buccal patch may be administered. In each case,dosing is such that the patient may administer a single dose.

Each dosage unit for oral administration contains suitably from 0.01 to500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula(I) or a pharmaceutically acceptable salt thereof, calculated as thefree base. The daily dosage for parenteral, nasal, oral inhalation,transmucosal or transdermal routes contains suitably from 0.01 mg to 100mg/Kg, of a compound of Formula(I). A topical formulation containssuitably 0.01 to 5.0% of a compound of Formula (I). The activeingredient may be administered from 1 to 6 times per day, preferablyonce, sufficient to exhibit the desired activity, as is readily apparentto one skilled in the art.

As used herein, “treatment” of a disease includes, but is not limited toprevention, retardation and prophylaxis of the disease.

Diseases and disorders which might be treated or prevented, includeimmune and inflammation-related diseases or disorders such as rheumatoidarthritis, Alzheimer's disease, psoriasis, gout, multiple sclerosis,systemic lupus erythematosus, glomerulonephritis and adult respiratorydistress syndrome.

Composition of Formula (I) and their pharmaceutically acceptable saltswhich are active when given orally can be formulated as syrups, tablets,capsules and lozenges. A syrup formulation will generally consist of asuspension or solution of the compound or salt in a liquid carrier forexample, ethanol, peanut oil, olive oil, glycerine or water with aflavoring or coloring agent. Where the composition is in the form of atablet, any pharmaceutical carrier routinely used for preparing solidformulations may be used. Examples of such carriers include magnesiumstearate, terra alba, talc, gelatin, acacia, stearic acid, starch,lactose and sucrose. Where the composition is in the form of a capsule,any routine encapsulation is suitable, for example using theaforementioned carriers in a hard gelatin capsule shell. Where thecomposition is in the form of a soft gelatin shell capsule anypharmaceutical carrier routinely used for preparing dispersions orsuspensions may be considered, for example aqueous gums, celluloses,silicates or oils, and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of acompound or salt in a sterile aqueous or non-aqueous carrier optionallycontaining a parenterally acceptable oil, for example polyethyleneglycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

Typical compositions for inhalation are in the form of a solution,suspension or emulsion that may be administered as a dry powder or inthe form of an aerosol using a conventional propellant such asdichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent, forexample polymeric glycols, gelatins, cocoa-butter or other low meltingvegetable waxes or fats or their synthetic analogs.

Typical dermal and transdermal formulations comprise a conventionalaqueous or non-aqueous vehicle, for example a cream, ointment, lotion orpaste or are in the form of a medicated plaster, patch or membrane.

Preferably the composition is in unit dosage form, for example a tablet,capsule or metered aerosol dose, so that the patient may administer asingle dose.

No unacceptable toxological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

The biological activity of the compounds of Formula (I) are demonstratedby the tests indicated hereinbelow.

Stable Expression of C3A Receptor in RBL-2H3 Cells

To prepare C3A receptor for expression in mammalian cells, a 1.6 kb cDNAfragment was obtained by PCR amplification that encompassed the entireC3A Receptor open reading frame. This fragment was subcloned intoKpnI/Hind III sites of the mammalian expression vector, pCDN (Aiyar, N.,et al (1994) Mol. Cell. Bio. 131, 75-86). Oligonucleotide primers usedfor PCR amplification were 5′-GA AGT GGT ACC ATG GCG TC-3′ and 5′-GC TCCAAG CTT TCA CAC AGT TG-3′ (the translation start and stop codons areunderlined). RBL-2H3 cells were electroporated with C3A in the pCDNmammalian expression vector (Aiyar, N., et al (1994) Mol. Cell. Bio.131, 75-86), exactly as described (DeMartino, J. A., et al (1994) J.Biol. Chem. 269, 14446-14450). Individual G418 resistant (400 μg/ml)colonies were isolated and expanded. Clonal cell lines expressing C3Areceptor, as determined by ability of the cell line to respond to C3A ina calcium mobilization assay, were chosen for further functional andbinding studies.

Preparation of Membranes

RBL-2H3 cells expressing the human C3A receptor (hC3AR) were cultured toconfluency at 37° C. in a humidified incubator with 5% CO2/95% air, inEarls MEM supplemented with non-essential amino acids, 10% fetal calfserum and 400 μg/ml G418. Although this cell line is normally adherent,nonadherent cells are always present in cultures. The nonadherent cellswere adapted to grow in suspension. Nonadherent cells from three T-150flasks were centrifuged at 1,000×g for 10 min and resuspended in 50-mlof the above medium in a 250 ml shake flask and over 7-10 days the cellswere expanded to 2.51 in a spinner flask. Cells were harvested bycentrifugation, 1,000×g for 10 min at 4° C., and membranes were isolatedusing a modification of the procedure of Ross et al., (1977). Briefly,the cell pellet was washed with PBS and resuspended in 30 ml ofhypotonic membrane buffer (20 mM Tris, pH 7.5, 2 mM MgCl₂, 0.1 mM EDTA,1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin, 1 μMpepstatin A) and incubated on ice for 5 min. The cell suspension washomogenized in 40 ml Dounce homogenizer and centrifuged at 1,000×g for15 min to remove nuclei and cellular debris. Cell membranes werepelleted at 100,000×g for 30 min at 4° C. Membranes were resuspended inmembrane buffer with 10% sucrose and layered over membrane buffer with40% sucrose and centrifuged at 100,000×g for 90 min at 4° C. Membranesat the interface were isolated and collected by centrifugation at100,000×g for 30 min. The membrane pellet was resuspended in 5.0 ml ofmembrane buffer and aliquots stored at −80° C. Protein concentration wasquantified using the BCA protein assay reagent (Pierce, Rockford, Ill.).

Scintillation Proximity Assay

All assays are performed in a 96-well micro-titre plate format. The96-well plates (1450-401) are obtained from Wallac, Turku, Finland.Human anaphylatoxin C3A was obtained from Advanced ResearchTechnologies, San Diego, Calif. with Bolton-Hunter custom iodinationbeing performed by NEN Research Products, Boston, Mass. with specificactivity of 2200 Ci/mmol. Wheatgerm agglutinin SPA (ScintillationProximity Assay) beads were obtained from Amersham Corp., ArlingtonHeights, Ill. The binding buffer consists of 20 mM Bis-Trispropane, 25mM NaCl, 1 mM MgSO₄, 0.1 mM EDTA at pH 8.0. Each reaction mixturecontains: 125I C3A (25 pM, obtained from NEN, Boston Mass.), wheatgermagglutinin SPA beads (0.1 mg), 0.35 μgs of RBL-2H3 C3A receptormembranes (this may vary with quality of membrane preparation), 23 ug/mlBSA and 0.03% CHAPS in binding buffer.

The membranes were prebound to SPA beads for 30 minutes on ice whileshaking. The mixture of membranes and beads were centrifuged for threeminutes at 2000 rpm. The supernatant was removed and the pellet wasresuspended to original volume in binding buffer with 50 ug/mL BSA.Samples of interest were dissolved in neat DMSO to yield a 20× solutionfollowed by a 1:1 mixture with H₂O to yield a 10×, 50% DMSO workingsolution. The order of addition was 10 uLs sample, 45 uLs membrane boundSPA beads followed by 45 uLs of radiolabled ligand in binding buffercontaining 0.06% CHAPS. The plates were covered with plate sealers fromDynex Technologies, Inc, and shaken for 20 minutes and incubated anadditional 40 minutes at room temperature. The plates were thencentrifuged for three minutes at 2000 rpm followed by counting on theWallac 1450 Micro Beta Plus Liquid Scintillation counter.

Calcium Functional Assays

7TM receptors which are expressed in HEK 293 cells have been shown to becoupled functionally to activation of PLC and calcium mobilizationand/or cAMP stimulation or inhibition. Basal calcium levels in the HEK293 cells in receptor-transfected or vector control cells were observedto be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressingrecombinant receptors are loaded with fura 2 and in a single day >150selected ligands or tissue/cell extracts are evaluated for agonistinduced calcium mobilization. Agonists presenting a calcium transientare tested in vector control cells to determine if the response isunique to the transfected cells expressing receptor.

Calcium Mobilization: C3a-induced Response in RBL-2H3 cells carrying C3areceptor:

Bioassays:

The functional activity of an antagonist of the C3a receptor isdemonstrated using the C3a-induced Ca²⁺ mobilization in RBL-2H3 cellsstably expressing C3a (RBL-2H3-C3a).

RBL-2H3-C3a Cell Culture Conditions:

RBL-2H3-C3a cells were cultured to near confluence in T-150 flasks at37°C. in a humidified incubator with 5% CO₂/95% air in Earls MEM withEarls salts (Gibco) supplemented with non-essential amino acids andL-glutamate, with 10% fetal calf serum (Gibco) and 400 ug/ml G418(Gibco).

Fluorescent Measurements-Calcium Mobilization:

The functional assay used to assess antagonist activity of compounds wasC3a-induced calcium mobilization in intact RBL-2H3-C3a cells. Cells werewashed with 50 mM Tris, pH 7.4 containing 1 mM EDTA. The [Ca²⁺]_(i) wasestimated with the calcium fluorescent probe-fura 2 (Grynkiewicz, etal., J. Biol. Chem., 1985, 260, 3440-3450). The media was aspirated fromRBL-2H3-C3a cells that were near confluence in T-150 flasks then 40 mlin Krebs Ringer Hensilet containing 0.1% BSA, 1.1 mM MgCl₂ and 5 mMHEPES, pH 7.4 (buffer A) was added. The diacetoxymethoxy ester of fura 2(fura 2/AM) was added at a concentration of 2 μM and incubated for 45min at 37° C. Buffer A was aspirated off the RBL-2H3-C3a cells and 40 mlof Buffer A was added to the cells and incubated for an additional 20min to allow complete hydrolysis of the entrapped ester. Buffer A wasaspirated and cells covered with ˜5 ml of Delbeccos Phosphate BufferedSaline with 1 mM EDTA (no calcium or magnesium) for 5 min at 37° C.Buffer is aspirated off and 40 ml of buffer A added to the cells whichwere then mechanically detached from the flasks. RBL-2H3-C3a cells weremaintained at room temperature until used in the fluorescent assay whichwas performed within 3 hours.

The fluorescence of fura 2 containing cells was measured with afluorometer designed by the Johnson Foundation BiomedicalInstrumentation Group. The fluorometer was equipped with a temperaturecontrol and a magnetic stirrer under the cuvette holder. Wavelengthswere set at 340 nm (10 nm band width) for excitation and 510 nm (20 nmband width) for emission. All experiments were performed at 37° C. withconstant stirring. For compound studies, fura 2 loaded cells werecentrifuged and resuspended in buffer A containing 1 mM CaCl₂ minus BSAat 10⁶ cells/mL. For assessment of agonist activity, a 2 mL aliquot ofRBL-2H3-C3a cells was added to a cuvette and warmed in a water bath to37° C. The 1 cm² cuvette was transferred to the fluorometer andfluorescence was recorded for 15 seconds to ensure a stable baselinebefore addition of compound. Fluorescence was recorded continuously forup to 2 mins after addition of compounds to monitor for the presence ofany agonist activity.

For antagonist studies, varying concentrations of compounds or vehiclewere added to the fura 2 loaded RBL-2H3-C3a cells and monitored for 1min to ensure that there was no change in baseline fluorescence followedby the addition of 1 nM C3a. The maximal [Ca²⁺]/fura 2 fluorescence wasthen determined for each sample. The [Ca²⁺]_(i) was calculated using thefollowing formula:$\left\lbrack {Ca}^{2 +} \right\rbrack_{i} = {224\quad ({nM})\frac{F - F_{\min}}{F_{\max} - F}}$

The percent of maximal C3a (1 nM) induced [Ca²⁺]_(i) was determined foreach concentration of compound and the IC50 defined as the concentrationof test compound that inhibits 50% of the maximal C3a response.Concentration response curves (5-7 concentrations) were run.

High-Throughput-Screening-Calcium Assay:

The calcium assay described above was converted to ahigh-throughput-screen (HTS) with the use of a 96 well FluorescentImaging Plate Reader (FLIPR) from Biomolecular Devices. This technologyallows the measurement of the intracellular calcium mobilization incells attached to the bottom of a 96 well plate.

For this procedure, cells were obtained from the T-150 flasks asdescribed above. The cells were plated into the 96 well plate at 30,000cells/well. With incubation in a humidified environment in a cellincubator at 37° C. for 18-24 hours, the cells attached to the bottomsurface of the 96 well plate.

The FLIPR works best with the visible wavelength calcium indicators,Fluo-3 and Calcium green-1. Both of these dyes have been usedsuccessfully for the HTS assay, but Fluo-3 was generally used. Typically4 uM Fluo-3 was loaded into the cells for 1 hr at 37° C. in cell mediawithout fetal calf serum and with 1.5 mM sulfinpyrazone to inhibit dyerelease from the cells. The media is aspirated from the cells and freshmedia was added for 10 min at 37° C. to allow hydrolysis of the dye andremove extracellular dye. The media was aspirated and replaced with KRHbuffer (buffer A above). After 10 min at 37° C. the cells were placed inFLIPR apparatus for analysis.

FLIPR has 3-96 well plates. In addition to the plate with dye loadedcells, there is a plate containing varying concentrations of compound orvehicle and the third plate has the agonist at varying concentrations toestablish agonist potency or a single concentration, e.g., 1 nM of C3afor antagonist activity. For antagonist studies, FLIPR obtains abaseline fluorescence for ˜30 sec, then it adds the compounds to all 96wells simultaneously and begins to monitor changes in intracellularCa²⁺. After 2 min, the contents or the agonist plate is added to thecells. The maximal Ca²⁺ response (in optical units) for 1 nM C3a in thepresence of vehicle (100%) or the various concentrations of compound isdetermined. Inhibition curves were generated essentially as describedfor the single cuvette Fura-2 assay described above.

The following examples are illustrative of the present invention but notintended to be limiting in any way.

EXAMPLE 1 (2-Naphthyloxy)acetylarginine

a) Fmocarginine(Boc)₂ Chlorotrityl Resin

To a mixture of Fmocarginine(Boc), (12.2 g) and chlorotrityl resin (1.8g, loading=1.05 mmol/g) in 30 mL of CH₂Cl₂ was addeddiisopropylethylamine (700 uL). The mixture was agitated for 16 h andthe liquid phase was drained. The resulting resin was washed withN-methylpyrollidine (2×) and CH₂Cl₂ (3×) to yield 2.2 g of product.

b) Bromoacetylarginine(Boc)₂ Chlorotrityl Resin

To Fmocarginine(Boc)²⁻Chlorotrityl resin (2.2 g) was added 20%piperidine in CH₂Cl₂ (50 mL). The mixture was agitated for 1 h and theliquid phase was drained. The resulting resin was washed with CH₂Cl₂(5×).

The resin was swelled in DMF and to the mixture was added bromoaceticacid (5.8 g) and EDC (2.9 g). The solution was agitated for 4 h and theliquid phase was drained. The resulting resin was washed withN-methylpyrollidine (2×) and CH₂Cl₂ (4×).

c) 2-Naphthyloxyacetylarginine(Boc)₂Chlorotrityl Resin

To bromoacetylarginine(Boc)²⁻Chlorotrityl resin (75 mg) in 1.5 mL ofDMSO was added 2-naphthol (10 eq) and potassium carbonate (100 mg), andthe mixture was shaken at 80° for 4 h. The liquid phase was drained andthe resin was washed with N-methylpyrollidine (2×) and CH₂Cl₂ (3×).

d) 2-Naphthyloxyacetylarginine

2-Naphthyloxyacetylarginine(Boc)²⁻Chlorotrityl resin (˜75 mg) wasagitated in 5 mL of 2.5% triisopropylsilane (TIS) in 1:1 CH₂Cl₂/TFA for2 h. The liquid phase was collected in a flask and the solvent wasevaporated under reduced pressure. The residue was dissolved in 1 mL ofTFA and added to 7 mL of ether to precipitate out the product as a whitesolid. The heterogeneous mixture was centrifuged and the solvent wasdecanted. Ether was added to the resulting solid, the mixturecentrifuged and the solvent decanted again. The product was dried undervacuum. ES(+) MS m/e=359.2 (M+H).

EXAMPLE 2 1-Naphthyloxyacetylarginine

a) Fmocarginine(Mtr) Wang

To Wang resin (10 g, 12 mmol) in CH₂Cl₂ (250 mL) was addedFmocarginine(Mtr) (9.31 g, 15 mmol), EDC (2.93 g, 15 mmol), and DMAP(1.44 g, 12 mmol). Argon was bubbled through it to agitate the reactionovernight. The slurry was filtered and washed with CH₂Cl₂ for 30 min.Solvent was drained off, and a second washing was performed at the samecondition for 5 h. Then the slurry was filtered, washed with CH₂Cl₂(1×), NMP (3×), CH₂Cl₂ (3×), and dried under high vaccum for 24 h togive Fmocarginine(Mtr) Wang. Nitrogen analysis indicated a substitutionof 0.73 mmol/g.

b) 1-Naphthyloxyacetylarginine(Mtr) Wang

Deprotection of Fmocarginine(Mtr) Wang was effected with 20% piperidinein CH₂Cl₂ (200 mL) for 30 min. The solution phase was drained, and asecond round of deprotection was carried out for 15 min. The solutionwas drained, and the resin was washed with CH₂Cl₂ (5×) to affordarginine(Mtr) Wang. To this intermediate (2 g, 2.4 mmol) in DMF (20 mL)was added 1-naphthyloxyacetic acid (2.43 g, 12 mmol), EDC (2.3 g, 12mmol), and HOBT (1.62 mmol, 12 mmol). After overnight shaking, theslurry was filtered, and washed with DMF (2×)/CH₂Cl₂ (6×).

c) 1-Naphthyloxyacetylarginine

To 1-naphthyloxyacetylarginine(Mtr) Wang was added 2.5% TIS in TFA (45mL). After shaking for 5 h, the solvent was evaporated under reducedpressure. The residue was purified by preparative HPLC to afford thetitle compound. ES(+) MS m/e=359.4 (M+H).

EXAMPLE 3 1-[7-(4-Hydroxyphenylmethyl)Naphthyloxy]acetylarginine

The title compound was obtained as a byproduct in example 2c). ES(+) MSm/e=465.4 (M+H).

EXAMPLE 4 (2,2-Diphenylethoxy)acetylarginine

a) Fmocaroinine(Boc)₂ Wang

To Fmocarginine(Boc)₂(1.8 g, 3 mmol) and Wang resin (2 g, 2 mmol) inCH₂Cl₂ (40 mL) was added EDC (573 mg, 3 mmol) and DMAP (244 mg, 2 mmol).The mixture was shaken overnight and washed with DMF (2×)/CH₂Cl₂ (6×).

b) (2,2-Diphenylethoxy)acetic Acid

To 2,2-diphenylethanol (1 g) in DMF (10 mL) was added 60% sodium hydride(350 mg) at 0° C. under Ar. The solution was stirred for 10 min, andt-butyl bromoacetate (888 uL) was added, and the solution was warmed toRT. After stirring 30 min, the reaction was quenched with water (20 mL),and the aqueous solution was extracted with ether (25 mL). The organiclayer was washed with water (20 mL) and brine (20 mL). The organicsolution was dried (MgSO₄), and silica gel flash chromatography (3%ethyl acetate/hexanes) yielded t-butyl (2,2-diphenylethoxy)acetate. Theintermediate was treated with 25% TFA/CH₂Cl₂ for 1 h. The solvent wasremoved, and traces of TFA was removed by azeotroping with toluene toyield the title compound. 1H NMR (CDCl₃) δ 7.1-7.4 (m, 10H), 4.32 (t,J=8.4 Hz, 1H), 4.0-4.1 (m, 4H).

c) (2,2-Diphenylethoxc)acetylarginine(Boc)₂ Wang

Fmocarginine(Boc)²⁻Wang (200 mg) was treated with 20% piperidine inCH₂Cl₂ (5 mL) for 30 min. The solvent was drained and the resin waswashed with CH₂Cl₂ (6×). To the resin in DMF (3.5 mL) was added(2,2-diphenylethoxy)acetic acid (92 mg), EDC (69 mg), and HOBT (49 mg),and the mixture was shaken overnight. The solution was drained, and theresin was washed with DMF (2×)/CH₂Cl₂ (6×).

d) (2,2-Diphenylethoxn)acetylarginine

The resin was treated with a solution of 2.5% TIS in 1:1 TFA/CH₂Cl₂ for90 min. The cleavage solution was collected, the solvent removed underreduced pressure, and traces of TFA were removed by azeotroping withtoluene. The residue was washed with hexanes (2×) to yield the titlecompound. ES(+) MS m/e=413.3 (M+H).

EXAMPLE 5 (3-Chlorobenzyloxy)acetylarginine

The tide compound was prepared according to the procedure of Example 4except substituting 3-chorobenzylalcohol for 2,2-diphenylethanol. ES(+)MS m/e=357.2 (M+H).

EXAMPLE 6 (2,2-Diphenylethoxy)acetyl-Nα-methylarginine

a) Nα-methylarginine(Mtr)OMe TFA Salt

To Boc-Nα-methylarginine(Mtr) (1 g, 2 mmol) in dry THF (50 mL) at 0° C.is added diazomethane (5 mmol) in ether (10 mL). The solution wasstirred for 2 h, and quenched with acetic acid. Ethyl acetate (50 mL)was added, the solution was washed with 10% sodium hydroxide and brine,and the organic layer was dried (MgSO₄).

Boc-Nα-methylarginine(Mtr)OMe was dissolved in 1:1 TFA/at 0° C., andstirred, with warming to RT for 2 h. When the reaction appeared completeas judged by TLC, the solvent was removed under reduced pressure. TraceTFA was removed by azeotroping with toluene. ES(+) MS m/e=415.4 (M+H).

b) (2,2-Diphenylethoxn)acetyl-Nα-methylarginine(Mtr)OMe

To Nα-methylarginine(Mtr)OMe TFA salt (0.188 g, 0.36 mmol) in 2 mL ofDMF was added DIEA (0.078 g, 0.60 mmol). Then a premixed solution of2,2-diphenylethoxyacetic acid (0.100 g, 0.39 mmol), PyBOP (0.370 g, 0.71mmol) and DIEA (0.196 g, 1.51 mmol) in 1 mL of DMF was added. Thesolution was stirred at RT overnight. Ethyl acetate was added, thesolution was washed with 3N HCl, brine, NaHCO₃(sat'd), and brine again.The organic layer was dried (MgSO₄), filtered, and the solvent wasremoved under reduced pressure. Purification by silica gelchromatography (50% ethyl acetate/hexanes) yielded the title compound(86 mg). ES(+) MS m/e=653.3 (M+H).

c) (2,2-Diphenylethoxy)acetyl-Nα-methylarginine(Mtr)

To (2,2-diphenylethoxy)acetyl-Nα-methylarginine(Mtr)OMe was added 2 mLof THF and 2 mL of 1M LiOH. The solution was stirred at RT for 2.5 h,ethyl acetate was added, and the aqueous layer was acidified with 3NHCl. The organic layer was dried (MgSO₄), filtered, and the solvent wasremoved under reduced pressure. Purification by silica gelchromatography (0.1% AcOH/2% MeOW/CH₂Cl₂) yielded the title compound (40mg). ES(+) MS m/e=639.2 (M+H).

d) (2,2-Diphenylethoxyacetyl-Nα-methylarginine

To (2,2-diphenylethoxy)acetyl-Nα-methylarginine(Mtr) (0.040 g, 0.06mmol) was added 2 mL of a 2.5% TIPS in 1:1 CH₂Cl₂/TFA solution. Thesolution was stirred at RT for 4 h. The solvent was removed underreduced pressure, and the title compound was precipitated out ofsolution using diethyl ether. ES(+) MS m/e=427.3 (M+H).

EXAMPLE 7

Inhalant Formulation

A compound of Formula (I), (1 mg to 100 mg) is aerosolized from ametered dose inhaler to deliver the desired amount of drug per use.

EXAMPLE 8

Tablet Formulation Tablets/Ingredients Per Tablet 1. Active ingredient40 mg (Cpd of Form. (I) 2. Corn Starch 20 mg 3. Alginic acid 20 mg 4.Sodium Alginate 20 mg 5. Mg stearate 1.3 mg 

Procedure for Tablet Formulation:

Ingredients 1, 2, 3 and 4 are blended in a suitable mixer/blender.Sufficient water is added portion-wise to the blend with careful mixingafter each addition until the mass is of a consistency to permit itsconversion to wet granules. The wet mass is converted to granules bypassing it through an oscillating granulator using a No. 8 mesh (2.38mm) screen. The wet granules are then dried in an oven at 140° F. (60°C.) until dry. The dry granules are lubricated with ingredient No. 5,and the lubricated granules are compressed on a suitable tablet press.

EXAMPLE 9

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of formula I inpolyethylene glycol with heating. This solution is then diluted withwater for injections (to 100 mL). The solution is then rendered sterileby filtration through a 0.22 micron membrane filter and sealed insterile containers.

All publications, including but not limited to patents and patentapplications cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference as though fullyset forth.

What is claimed is:
 1. A compound according to Formula (I):

wherein: A represents C₁₋₄ alkylene, unsubstituted or optionallysubstituted by C₁₋₄ alkyl or aryl; or A forms a 5-8 membered fusedaliphatic ring with the adjacent phenyl ring; m is an integer from 1 to3; each R₁ is independently selected form the group consisting of halo,C₁₋₄ alkyl, methanesulfonyl, alkoxy, nitrile, dimethylamine,methylenedioxy and CF₃; and R₂ is hydrogen or methyl.
 2. A compoundaccording to claim 1 wherein the asterisk*represents an S configuration.3. A compound according to claim 2 wherein A is phenethyl.
 4. A compoundaccording to claim 3 wherein R₁ is hydrogen.
 5. A compound according toclaim 4 wherein m is
 0. 6. A compound according to claim 1 selected fromthe group consisting of:1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;(2,2-Diphenylethoxy)acetylarginine;(2,2-Diphenylethoxy)acetyl-Nα-methylarginine;(3-Chlorobenzyloxy)acetylarginine; 2-Naphthyloxyacetylarginine;(2,3-Dimethylphenoxy)acetylarginine; 8-(Quinolinyloxy)acetylarginine;6-(Quinolinyloxy)acetylarginine; 2-(1-Bromonaphthyloxy)acetylarginine;(4-Benzyloxyphenoxy)acetylarginine; and2-(6-Methoxynaphthyloxy)acetylarginine.
 7. A compound according to claim6 selected from the group consisting of:1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;(2,2-Diphenylethoxy)acetylarginine; and 2-Naphthyloxyacetylarginine. 8.(2,2-Diphenylethoxy)acetylarginine.
 9. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 10. A method of antagonizing a C3A receptor which comprisesadministering to a subject in need thereof, an effective amount of acompound of claim
 1. 11. A method according to claim 10 wherein thecompound is selected from the group consisting of:1-Naphthyloxyacetylarginine;1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;(2,2-Diphenylethoxy)acetylarginine;(2,2-Diphenylethoxy)acetyl-Nα-methylarginine;(3-Chlorobenzyloxy)acetylarginine; 2-Naphthyloxyacetylarginine;(2,3-Dimethylphenoxy)acetylarginine; 8-(Quinolinyloxy)acetylarginine;6-(Quinolinyloxy)acetylarginine; 2-(1-Bromonaphthyloxy)acetylarginine;(4-Benzyloxyphenoxy)acetylarginine; and2-(6-Methoxynaphthyloxy)acetylarginine.
 12. A method of treating animmune or inflammatory disease or disorder characterized by abnormallevels of anaphylatoxins which comprises administering to a subject inneed thereof an effective amount of a compound of claim
 1. 13. A methodaccording to claim 12 wherein the disease or disorder is selected fromthe group consisting of rheumatoid arthritis, Alzheimer's disease,psoriasis, gout, multiple sclerosis, systemic lupus erythematosus,glomerulonephritis and adult respiratory distress syndrome.